Potato transformation vector pSIM1278

ABSTRACT

A potato transformation vector, pSIM1278 is disclosed. The invention relates to the potato transformation vector for silencing endogenous aspargine synthetase-1, polyphenol oxidase-5, phosphorylase-L, and dikinase R1 genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Entry of International PatentApplication No. PCT/US2013/068543, filed on Nov. 5, 2013, which claimsthe benefit of priority from U.S. provisional patent application Ser.No. 61/818,752, filed on May 2, 2013, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a novel potato cultivar designated E12and to the tubers, plants, plant parts, tissue culture and seedsproduced by that potato variety. The invention further relates to foodproducts produced from potato cultivar E12, such as French fries, potatochips, dehydrated potato material, potato flakes and potato granules.All publications cited in this application are herein incorporated byreference.

The potato is the world's fourth most important food crop and by far themost important vegetable. Potatoes are currently grown commercially innearly every state of the United States. Annual potato productionexceeds 18 million tons in the United States and 300 million tonsworldwide. The popularity of the potato derives mainly from itsversatility and nutritional value. Potatoes can be used fresh, frozen ordried, or can be processed into flour, starch or alcohol. They containcomplex carbohydrates and are rich in calcium, niacin and vitamin C.

The quality of potatoes in the food industry is adversely affected bytwo critical factors: (1) potatoes contain large amounts of asparagine,a non-essential free amino acid that is rapidly oxidized to formacrylamide, a carcinogenic product, upon frying or baking; and (2)potatoes are highly susceptible to enzymatic browning and discoloration,an undesirable event which happens when polyphenol oxidase leaks outfrom the damaged plastids of bruised potatoes. In the cytoplasm, theenzyme oxidizes phenols, which then rapidly polymerize to produce darkpigments. Tubers contain large amounts of phosphorylated starch, some ofwhich is degraded during storage to produce glucose and fructose. Thesereducing sugars react with amino acids to form Maillard productsincluding acrylamide when heated at temperatures above 120° C. Twoenzymes involved in starch phosphorylation are water dikinase R1 andphosphorylase-L (R1 and PhL). Browning is also triggerednon-enzymatically as a consequence of the partial degradation of starchinto glucose and fructose.

To date, there are no potato plant varieties that produce tubers withlow acrylamide content, increased black spot bruise tolerance andreduced senescence sweetening. Thus, there is a need to develop potatovarieties with reduced levels of toxic compounds, but without the use ofunknown or foreign nucleic acids. The present invention satisfies thisneed.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope. In various embodiments, one or more ofthe above-described problems have been reduced or eliminated, whileother embodiments are directed to other improvements.

To this end, the present invention provides novel potato variety E12transformed with nucleic acid sequences that are native to the potatoplant genome and does not contain foreign DNA, Agrobacterium DNA, viralmarkers or vector backbone sequences. Rather, the DNA inserted into thegenome of the potato variety E12 is a non-coding polynucleotide nativeto potato or native to wild potato, a potato sexually-compatible plant,that silences genes involved in the expression of black spot bruises,asparagine accumulation and senescence sweetening.

Thus, in one embodiment, the present invention provides a plant vector,referred to as pSIM278, that comprises a first silencing cassettecontaining two copies of a DNA segment comprising, in anti-senseorientation, a fragment of the asparagine synthetase-1 gene (fAsn1) andthe 3′-untranslated sequence of the polyphenol oxidase-5 gene; and asecond silencing cassette containing two copies of a DNA segmentcomprising, in anti-sense orientation, a fragment of the potatophosphorylase-L (pPhL) gene and a fragment of the potato R1 gene. ThepSIM1278 vector comprises a 9,511 bp backbone region that supportsmaintenance of the plant DNA prior to plant transformation and is nottransferred into plant cells upon transformation of the plant cells, anda 10,147 bp DNA insert region comprising native DNA that is stablyintegrated into the genome of the plant cells upon transformation.

In a different embodiment, the invention provides a plant celltransformed with the plant vector of the invention. In a furtherembodiment, the invention provides a potato plant variety comprising oneor more cells transformed with the vector of the invention. In oneaspect of the invention, the potato plant variety expresses at least oneof the two silencing cassettes of the vector, and expression of thesilencing cassette results in the down-regulation of the asparaginesynthetase-1 gene and the polyphenol oxidase-5 gene in the tubers of theintragenic plant. In a preferred aspect of the invention, the tubers ofthe potato plant variety expressing at least one silencing cassettedisplay two or more desirable traits that are not present in the tubersof untransformed plants of the same variety. In the most preferredaspect of the invention, the two or more desirable traits are selectedfrom the group consisting of low asparagine accumulation, reducedblack-spot bruising and reduced heat-induced acrylamide formation.

In a different aspect of the invention, the potato plant varietyexpresses both silencing cassettes of the plant DNA vector, andexpression of the silencing cassettes results in the down-regulation ofthe asparagine synthetase-1 gene, the polyphenol oxidase-5 gene, thephosphorylase-L gene and the dikinase R1 gene in the tubers of thepotato plant variety. In a preferred aspect of the invention, the tubersof the potato plant variety expressing two silencing cassettes of theplant DNA vector display two or more desirable traits that are notpresent in the tubers of untransformed plants of the same variety. In apreferred embodiment, the two or more desirable traits are selected fromthe group consisting of low asparagine accumulation, reduced black-spotbruising, reduced accumulation of reducing sugars during storage andreduced heat-induced acrylamide formation. In one aspect of theinvention, the potato plant variety expressing the two silencingcassettes of the plant DNA vector is the Russet Burbank E12 variety.

Thus, according to the invention, there is provided a new potatocultivar of the genus and species Solanum tuberosum L. designated E12.This invention thus relates to potato cultivar E12, to the tubers ofpotato cultivar E12, to the plants of potato cultivar E12, to the seedsof potato cultivar E12, to the food products produced from potatocultivar E12, and to methods for producing a potato plant produced byselfing potato cultivar E12 or by crossing potato cultivar E12 withanother potato cultivar, and the creation of variants by mutagenesis ortransformation of potato cultivar E12.

Thus, any such methods using the cultivar E12 are embodiments of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using potato cultivar E12as at least one parent are within the scope of this invention.Advantageously, the potato cultivar could be used in crosses with other,different, potato plants to produce first generation (F₁) potato hybridtubers, seeds and plants with superior characteristics.

In another embodiment, the present invention provides for single ormultiple gene converted plants of potato cultivar E12. In oneembodiment, the transferred gene(s) may be a dominant or recessiveallele(s). In some embodiments, the transferred gene(s) will confer suchtraits as herbicide resistance, insect resistance, resistance forbacterial, fungal, or viral disease, male fertility, male sterility,enhanced nutritional quality, uniformity, and increase in concentrationof starch and other carbohydrates, decrease in tendency to bruise anddecrease in the rate of conversion of starch to sugars. The gene(s) maybe a naturally occurring potato gene or a transgene introduced throughgenetic engineering techniques, backcrossing or mutation.

In another embodiment, the present invention provides regenerable cellsfor use in tissue culture of potato cultivar E12. In one embodiment, thetissue culture will be capable of regenerating plants having all thephysiological and morphological characteristics of the foregoing potatoplant, and of regenerating plants having substantially the same genotypeas the foregoing potato plant. In some embodiments, the regenerablecells in such tissue cultures will be embryos, protoplasts, meristematiccells, callus, pollen, leaves, anthers, pistils, cotyledons, hypocotyl,roots, root tips, flowers, seeds, petioles, tubers, eyes or stems. Stillfurther, the present invention provides potato plants regenerated fromtissue cultures of the invention.

In a further embodiment, the invention provides a food product made froma tuber of potato plant variety Russet Burbank E12. Preferably, the foodproduct is a heat-treated product. Even more preferably, the foodproduct is a French fry, potato chip, dehydrated potato material, potatoflakes, or potato granules.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the pSIM1278 transformation vector. The vector backboneregion, on the left, is 9,511 bp long, as it starts at position 9,957 bpand ends at position 19,468 bp. The backbone DNA consists mainly ofbacterial DNA which provides support maintenance of the DNA insert priorto plant transformation. The DNA insert region (right side) is 10,147 bplong (from 19,469 bp to 19,660 bp and from 1 bp to 9,956 bp). The DNAinsert consists of native DNA only and was stably integrated into thepotato genome upon transformation.

FIG. 2 provides a schematic representation of the silencing cassettes inthe DNA insert inserted in the pSIM1278 transformation vector. Eachsilencing cassette contains two copies of two gene fragments separatedby a spacer. Two copies of a DNA segment comprising fragments of fourtargeted genes, namely Asn-1, Ppo-5, Ph1 and R1, were inserted asinverted repeats between two convergent promoters, indicated as Pro,that are predominantly active in tubers. Plants containing the resultingsilencing cassette produce a diverse and unpolyadenylated array of RNAmolecules in tubers that dynamically and vigorously silence the intendedtarget genes. The size of the RNA molecules was generally smaller thanthe distance between the two promoters employed because convergenttranscription results in collisional transcription.

FIG. 3 shows the results of the catechol assay. The Russet Burbankcontrol is on the left and variety E12 is on the right. Dark brownindicates that the trait for black spot bruise reduction is not present.As seen in FIG. 3, E12 has the trait for black spot bruise reduction,whereas the control does not.

DETAILED DESCRIPTION OF THE INVENTION

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Amino acid sequence. As used herein, includes an oligopeptide, peptide,polypeptide, or protein and fragments thereof that are isolated from,native to, or naturally occurring in a plant, or are synthetically madebut comprise the nucleic acid sequence of the endogenous counterpart.

Artificially manipulated. as used herein, “artificially manipulated”means to move, arrange, operate or control by the hands or by mechanicalmeans or recombinant means, such as by genetic engineering techniques, aplant or plant cell, so as to produce a plant or plant cell that has adifferent biological, biochemical, morphological, or physiologicalphenotype and/or genotype in comparison to unmanipulated,naturally-occurring counterpart.

Asexual propagation. Producing progeny by generating an entire plantfrom leaf cuttings, stem cuttings, root cuttings, tuber eyes, stolons,single plant cells protoplasts, callus and the like, that does notinvolve fusion of gametes.

Backbone. Nucleic acid sequence of a binary vector that excludes the DNAinsert sequence intended for transfer.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Bacterial Ring Rot. Bacterial ring rot is a disease caused by thebacterium Clavibacter michiganense ssp. Bacterial ring rot derives itsname from a characteristic breakdown of the vascular ring within thetuber. This ring often appears as a creamy-yellow to light-brown, cheesyrot. On the outer surface of the potato, severely diseased tubers mayshow slightly sunken, dry and cracked areas. Symptoms of bacterial ringrot in the vascular tissue of infected tubers can be less obvious thandescribed above, appearing as only a broken, sporadically appearing darkline or as a continuous, yellowish discoloration.

Black spot bruise. Black spots found in bruised tuber tissue are aresult of a pigment called melanin that is produced following the injuryof cells and gives tissue a brown, gray or black appearance. Melanin isformed when phenol substrates and an appropriate enzyme come in contactwith each other as a result of cellular damage. The damage does notrequire broken cells. However, mixing of the substrate and enzyme mustoccur, usually when the tissue is impacted. Black spots occur primarilyin the perimedullary tissue just beneath the vascular ring, but may belarge enough to include a portion of the cortical tissue.

Border-like sequences. A “border-like” sequence is isolated from theselected plant species that is to be modified, or from a plant that issexually-compatible with the plant species to be modified, and functionslike the border sequences of Agrobacterium. That is, a border-likesequence of the present invention promotes and facilitates theintegration of a polynucleotide to which it is linked. A border-likesequence is between 5-100 bp in length, 10-80 bp in length, 15-75 bp inlength, 15-60 bp in length, 15-50 bp in length, 15-40 bp in length,15-30 bp in length, 16-30 bp in length, 20-30 bp in length, 21-30 bp inlength, 22-30 bp in length, 23-30 bp in length, 24-30 bp in length,25-30 bp in length, or 26-30 bp in length. Left and right bordersequences are isolated from and/or native to the genome of a plant thatis to be modified. A border-like sequence is not identical in nucleotidesequence to any known Agrobacterium-derived T-DNA border sequence. Thus,a border-like sequence may possess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides that aredifferent from a T-DNA border sequence from an Agrobacterium species,such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. That is,a border, or a border-like sequence of the present invention has atleast 95%, at least 90%, at least 80%, at least 75%, at least 70%, atleast 60% or at least 50% sequence identity with a T-DNA border sequencefrom an Agrobacterium species, such as Agrobacterium tumefaciens orAgrobacterium rhizogenes, but not 100% sequence identity. A border-likesequence can be isolated from a plant genome and be modified or mutatedto change the efficiency by which it is capable of integrating anucleotide sequence into another nucleotide sequence. Otherpolynucleotide sequences may be added to or incorporated within aborder-like sequence of the present invention. Thus, a left border or aright border may be modified so as to possess 5′- and 3′-multiplecloning sites, or additional restriction sites. A border sequence may bemodified to increase the likelihood that backbone DNA from theaccompanying vector is not integrated into the plant genome.

Consisting essentially of. A composition “consisting essentially of”certain elements is limited to the inclusion of those elements, as wellas to those elements that do not materially affect the basic and novelcharacteristics of the inventive composition. Thus, so long as thecomposition does not affect the basic and novel characteristics of theinstant invention, that is, does not contain foreign DNA that is notfrom the selected plant species or a plant that is sexually compatiblewith the selected plant species, then that composition may be considereda component of an inventive composition that is characterized by“consisting essentially of” language.

Cotyledon. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

Degenerate primer. A “degenerate primer” is an oligonucleotide thatcontains sufficient nucleotide variations that it can accommodate basemismatches when hybridized to sequences of similar, but not exact,homology.

Dicotyledon (dicot). A flowering plant whose embryos have two seedleaves or cotyledons. Examples of dicots include, but are not limitedto, tobacco, tomato, potato, sweet potato, cassava, legumes includingalfalfa and soybean, carrot, strawberry, lettuce, oak, maple, walnut,rose, mint, squash, daisy, and cactus.

DNA insert. According to the present invention, the DNA insert to beinserted into the genome of a plant comprises polynucleotide sequencesnative to that plant or has native genetic elements to that plant. Inone example, for instance, the DNA insert of the potato variety E12 ofthe present invention is a 10,147 bp non-coding polynucleotide that isnative to potato or wild potato, a potato sexually-compatible plant,that is stably integrated into the genome of the plant cells upontransformation and silences genes involved in the expression of blackspot bruises, asparagine accumulation and senescence sweetening. The DNAinsert preferably comprises two expression cassettes and is insertedinto a transformation vector referred to as the pSIM1278 transformationvector. The first cassette comprises fragments of both the asparaginesynthetase-1 gene (Asn1) and the polyphenol oxidase-5 gene (Ppo5),arranged as inverted repeats between the Agp promoter of the ADP glucosepyrophosphorylase gene (Agp) and the Gbss promoter of the granule-boundsynthase gene (Gbss). These promoters are predominantly active intubers. The function of the second cassette is to silence the promotersof the starch associated gene dikinase-R1 (R1) and the phosphorylase-Lgene (PhL). This cassette is comprised of fragments of the promoters ofthe starch associated gene dikinase-R1 (R1) and the phosphorylase-L gene(PhL), operably linked to the same Agp and Gbss promoters as the firstcassette. These expression cassettes contain no foreign DNA, and consistof DNA only from either the selected plant species or from a plant thatis sexually compatible with the selected plant species.

Embryo. The embryo is the immature plant contained within a mature seed.

Foreign. “Foreign,” with respect to a nucleic acid, means that thatnucleic acid is derived from non-plant organisms, or derived from aplant that is not the same species as the plant to be transformed or isnot derived from a plant that is not interfertile with the plant to betransformed, does not belong to the species of the target plant.According to the present invention, foreign DNA or RNA representsnucleic acids that are naturally occurring in the genetic makeup offungi, bacteria, viruses, mammals, fish or birds, but are not naturallyoccurring in the plant that is to be transformed. Thus, a foreignnucleic acid is one that encodes, for instance, a polypeptide that isnot naturally produced by the transformed plant. A foreign nucleic aciddoes not have to encode a protein product. According to the presentinvention, a desired intragenic plant is one that does not contain anyforeign nucleic acids integrated into its genome.

Gene. As used herein, “gene” refers to the coding region and does notinclude nucleotide sequences that are 5′- or 3′- to that region. Afunctional gene is the coding region operably linked to a promoter orterminator. A gene can be introduced into a genome of a species, whetherfrom a different species or from the same species, using transformationor various breeding methods.

Gene Converted (Conversion). Gene converted (conversion) plant refers toplants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe one or more genes transferred into the variety via the backcrossingtechnique, via genetic engineering or via mutation. One or more loci mayalso be transferred.

Genetic rearrangement. Refers to the re-association of genetic elementsthat can occur spontaneously in vivo as well as in vitro which introducea new organization of genetic material. For instance, the splicingtogether of polynucleotides at different chromosomal loci, can occurspontaneously in vivo during both plant development and sexualrecombination. Accordingly, recombination of genetic elements bynon-natural genetic modification techniques in vitro is akin torecombination events that also can occur through sexual recombination invivo. The insertion of a DNA insert into a plant genome, for instance,is an example of a genetic or genomic rearrangement.

Golden nematode. Globodera rostochiensis, commonly known as goldennematode, is a plant parasitic nematode affecting the roots and tubersof potato plants. Symptoms include poor plant growth, wilting, waterstress and nutrient deficiencies.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

In frame. Nucleotide triplets (codons) are translated into a nascentamino acid sequence of the desired recombinant protein in a plant cell.Specifically, the present invention contemplates a first nucleic acidlinked in reading frame to a second nucleic acid, wherein the firstnucleotide sequence is a gene and the second nucleotide is a promoter orsimilar regulatory element.

Integrate. Refers to the insertion of a nucleic acid sequence from aselected plant species, or from a plant that is from the same species asthe selected plant, or from a plant that is sexually compatible with theselected plant species, into the genome of a cell of a selected plantspecies. “Integration” refers to the incorporation of only nativegenetic elements into a plant cell genome. In order to integrate anative genetic element, such as by homologous recombination, the presentinvention may “use” non-native DNA as a step in such a process. Thus,the present invention distinguishes between the “use of” a particularDNA molecule and the “integration” of a particular DNA molecule into aplant cell genome.

Introduction. As used herein, refers to the insertion of a nucleic acidsequence into a cell, by methods including infection, transfection,transformation or transduction.

Isolated. “Isolated” refers to any nucleic acid or compound that isphysically separated from its normal, native environment. The isolatedmaterial may be maintained in a suitable solution containing, forinstance, a solvent, a buffer, an ion, or other component, and may be inpurified, or unpurified, form.

Leader. Transcribed but not translated sequence preceding (or 5′ to) agene.

Locus. A locus confers one or more traits such as, for example, malesterility, herbicide tolerance, insect resistance, disease resistance,waxy starch, modified fatty acid metabolism, modified phytic acidmetabolism, modified carbohydrate metabolism, and modified proteinmetabolism. The trait may be, for example, conferred by a naturallyoccurring gene introduced into the genome of the variety bybackcrossing, a natural or induced mutation, or a transgene introducedthrough genetic transformation techniques. A locus may comprise one ormore alleles integrated at a single chromosomal location.

Marketable Yield. Marketable yield is the weight of all tubers harvestedthat are between 2 and 4 inches in diameter. Marketable yield ismeasured in cwt (hundred weight) where cwt=100 pounds.

Monocotyledon (monocot). A flowering plant whose embryos have onecotyledon or seed leaf. Examples of monocots include, but are notlimited to turf grass, maize, rice, oat, wheat, barley, sorghum, orchid,iris, lily, onion, and palm.

Native. A “native” genetic element refers to a nucleic acid thatnaturally exists in, orginates from, or belongs to the genome of a plantthat is to be transformed. Thus, any nucleic acid, gene, polynucleotide,DNA, RNA, mRNA, or cDNA molecule that is isolated either from the genomeof a plant or plant species that is to be transformed or is isolatedfrom a plant or species that is sexually compatible or interfertile withthe plant species that is to be transformed, is “native” to, i.e.,indigenous to, the plant species. In other words, a native geneticelement represents all genetic material that is accessible to plantbreeders for the improvement of plants through classical plant breeding.Any variants of a native nucleic acid also are considered “native” inaccordance with the present invention. In this respect, a “native”nucleic acid may also be isolated from a plant or sexually compatiblespecies thereof and modified or mutated so that the resultant variant isgreater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%,76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,62%, 61%, or 60% similar in nucleotide sequence to the unmodified,native nucleic acid isolated from a plant. A native nucleic acid variantmay also be less than about 60%, less than about 55%, or less than about50% similar in nucleotide sequence. A “native” nucleic acid isolatedfrom a plant may also encode a variant of the naturally occurringprotein product transcribed and translated from that nucleic acid. Thus,a native nucleic acid may encode a protein that is greater than or equalto 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%,85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%,71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% similar inamino acid sequence to the unmodified, native protein expressed in theplant from which the nucleic acid was isolated.

Native genetic elements. “Native genetic elements” can be incorporatedand integrated into a selected plant species genome according to thepresent invention. Native genetic elements are isolated from plants thatbelong to the selected plant species or from plants that are sexuallycompatible with the selected plant species. For instance, native DNAincorporated into cultivated potato (Solanum tuberosum) can be derivedfrom any genotype of S. tuberosum or any genotype of a wild potatospecies that is sexually compatible with S. tuberosum (e.g., S.demissum).

Naturally occurring nucleic acid. Naturally occurring nucleic acid arefound within the genome of a selected plant species and may be a DNAmolecule or an RNA molecule. The sequence of a restriction site that isnormally present in the genome of a plant species can be engineered intoan exogenous DNA molecule, such as a vector or oligonucleotide, eventhough that restriction site was not physically isolated from thatgenome. Thus, the present invention permits the synthetic creation of anucleotide sequence, such as a restriction enzyme recognition sequence,so long as that sequence is naturally occurring in the genome of theselected plant species or in a plant that is sexually compatible withthe selected plant species that is to be transformed.

Operably linked. Combining two or more molecules in such a fashion thatin combination they function properly in a plant cell. For instance, apromoter is operably linked to a structural gene when the promotercontrols transcription of the structural gene.

Plant. As used herein, the term “plant” includes but is not limited toangiosperms and gymnosperms such as potato, tomato, tobacco, alfalfa,lettuce, carrot, strawberry, sugarbeet, cassava, sweet potato, soybean,maize, turf grass, wheat, rice, barley, sorghum, oat, oak, eucalyptus,walnut, and palm. Thus, a plant may be a monocot or a dicot. The word“plant,” as used herein, also encompasses plant cells, seed, plantprogeny, propagule whether generated sexually or asexually, anddescendents of any of these, such as cuttings or seed. Plant cellsinclude suspension cultures, callus, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,seeds and microspores. Plants may be at various stages of maturity andmay be grown in liquid or solid culture, or in soil or suitable media inpots, greenhouses or fields. Expression of an introduced leader, traileror gene sequences in plants may be transient or permanent. A “selectedplant species” may be, but is not limited to, a species of any one ofthese “plants.”

Plant Parts. As used herein, the term “plant parts” (or a potato plant,or a part thereof) includes but is not limited to protoplast, leaf,stem, root, root tip, anther, pistil, seed, embryo, pollen, ovule,cotyledon, hypocotyl, flower, tuber, eye, tissue, petiole, cell,meristematic cell, and the like.

Plant species. The group of plants belonging to various officially namedplant species that display at least some sexual compatibility.

Plant transformation and cell culture. Broadly refers to the process bywhich plant cells are genetically modified and transferred to anappropriate plant culture medium for maintenance, further growth, and/orfurther development.

Precise breeding. Refers to the improvement of plants by stableintroduction of nucleic acids, such as native genes and regulatoryelements isolated from the selected plant species, or from another plantin the same species as the selected plant, or from species that aresexually compatible with the selected plant species, into individualplant cells, and subsequent regeneration of these genetically modifiedplant cells into whole plants. Since no unknown or foreign nucleic acidis permanently incorporated into the plant genome, the inventivetechnology makes use of the same genetic material that is alsoaccessible through conventional plant breeding.

Progeny. As used herein, includes an F₁ potato plant produced from thecross of two potato plants where at least one plant includes potatocultivar E12 and progeny further includes, but is not limited to,subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, and F₁₀ generational crosseswith the recurrent parental line.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Recombinant. As used herein, broadly describes various technologieswhereby genes can be cloned, DNA can be sequenced, and protein productscan be produced. As used herein, the term also describes proteins thathave been produced following the transfer of genes into the cells ofplant host systems.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Regulatory sequences. Refers to those sequences which are standard andknown to those in the art, that may be included in the expressionvectors to increase and/or maximize transcription of a gene of interestor translation of the resulting RNA in a plant system. These include,but are not limited to, promoters, peptide export signal sequences,introns, polyadenylation, and transcription termination sites. Methodsof modifying nucleic acid constructs to increase expression levels inplants are also generally known in the art (see, e.g. Rogers et al., 260J. Biol. Chem. 3731-38, 1985; Cornejo et al., 23 Plant Mol. Biol. 567:81, 1993). In engineering a plant system to affect the rate oftranscription of a protein, various factors known in the art, includingregulatory sequences such as positively or negatively acting sequences,enhancers and silencers, as well as chromatin structure may have animpact. The present invention provides that at least one of thesefactors may be utilized in engineering plants to express a protein ofinterest. The regulatory sequences of the present invention are nativegenetic elements, i.e., are isolated from the selected plant species tobe modified.

Selectable marker. A “selectable marker” is typically a gene that codesfor a protein that confers some kind of resistance to an antibiotic,herbicide or toxic compound, and is used to identify transformationevents. Examples of selectable markers include the streptomycinphosphotransferase (spt) gene encoding streptomycin resistance, thephosphomannose isomerase (pmi) gene that converts mannose-6-phosphateinto fructose-6 phosphate; the neomycin phosphotransferase (nptII) geneencoding kanamycin and geneticin resistance, the hygromycinphosphotransferase (hpt or aphiv) gene encoding resistance tohygromycin, acetolactate synthase (als) genes encoding resistance tosulfonylurea-type herbicides, genes coding for resistance to herbicideswhich act to inhibit the action of glutamine synthase such asphosphinothricin or basta (e.g., the bar gene), or other similar genesknown in the art.

Sense suppression. Reduction in expression of an endogenous gene byexpression of one or more an additional copies of all or part of thatgene in transgenic plants.

Specific gravity. As used herein, “specific gravity” is an expression ofdensity and is a measurement of potato quality. There is a highcorrelation between the specific gravity of the tuber and the starchcontent and percentage of dry matter or total solids. A higher specificgravity contributes to higher recovery rate and better quality of theprocessed product.

T-DNA-Like. A “T-DNA-like” sequence is a nucleic acid that is isolatedfrom a selected plant species, or from a plant that is sexuallycompatible with the selected plant species, and which shares at least75%, 80%, 85%, 90%, or 95%, but not 100%, sequence identity withAgrobacterium species T-DNA. The T-DNA-like sequence may contain one ormore border or border-like sequences that are each capable ofintegrating a nucleotide sequence into another polynucleotide.

Total Yield. Total yield refers to the total weight of all harvestedtubers.

Trailer. Transcribed but not translated sequence following (or 3′to) agene.

Transcribed DNA. DNA comprising both a gene and the untranslated leaderand trailer sequence that are associated with that gene, which istranscribed as a single mRNA by the action of the preceding promoter.

Transformation of plant cells. A process by which DNA is stablyintegrated into the genome of a plant cell. “Stably” refers to thepermanent, or non-transient retention and/or expression of apolynucleotide in and by a cell genome. Thus, a stably integratedpolynucleotide is one that is a fixture within a transformed cell genomeand can be replicated and propagated through successive progeny of thecell or resultant transformed plant. Transformation may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion ofnucleic acid sequences into a prokaryotic or eukaryotic host cell,including Agrobacterium-mediated transformation protocols, viralinfection, whiskers, electroporation, heat shock, lipofection,polyethylene glycol treatment, micro-injection, and particlebombardment.

Transgene. A gene that will be inserted into a host genome, comprising aprotein coding region. In the context of the instant invention, theelements comprising the transgene are isolated from the host genome.

Transgenic plant. A genetically modified plant which contains at leastone transgene.

Variant. A “variant,” as used herein, is understood to mean a nucleotideor amino acid sequence that deviates from the standard, or given,nucleotide or amino acid sequence of a particular gene or protein. Theterms, “isoform,” “isotype,” and “analog” also refer to “variant” formsof a nucleotide or an amino acid sequence. An amino acid sequence thatis altered by the addition, removal or substitution of one or more aminoacids, or a change in nucleotide sequence, may be considered a “variant”sequence. The variant may have “conservative” changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. A variant may have“nonconservative” changes, e.g., replacement of a glycine with atryptophan. Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted may be foundusing computer programs well known in the art such as Vector NTI Suite(InforMax, MD) software.

Vine Maturity. Vine maturity refers to a plant's ability to continue toutilize carbohydrates and photosynthesize. Vine maturity is scored on ascale of 1 to 5 where 1=dead vines and 5=vines green, still flowering.

The insertion of desirable traits into the genome of potato plantspresents particular difficulties because potato is tetraploid, highlyheterozygous and sensitive to in-breeding depression. It is thereforevery difficult to efficiently develop transgenic potato plants thatproduce less acrylamide and less harmful Maillard-reaction products,including N-Nitroso-N-(3-keto-1,2-butanediol)-3′-nitrotyramine (Wang etal., Arch Toxicol 70: 10-5, 1995), 5-hydroxymethyl-2-furfural (Janzowskiet al., Food Chem Toxicol 38: 801-9, 2000), and other Maillard reactionproducts with mutagenic properties (Shibamoto, Prog Clin Biol Res 304:359-76, 1989), during processing using conventional breeding.

Several methods have been tested and research is ongoing to reduceacrylamide through process changes, reduction in dextrose, and additivessuch as asparaginase, citrate, and competing amino acids. The requiredcapital expense to implement process changes throughout the potatoindustry would cost millions of dollars. In addition to the expense,these process changes have significant drawbacks including potentiallynegative flavors associated with additives such as asparaginase orcitrate. Typically, fry manufacturers add dextrose during processing offrench fries to develop the desired golden brown color, but dextrosealso increases the formation of acrylamide through the Maillardreaction. Significant reductions in acrylamide occur by merely omittingdextrose from the process; however, the signature golden brown colorsmust then be developed some other way (such as though the addition ofcolors like annatto) The use of alternate colors, results in an absenceof the typical flavors that develop through those browning reactions.Another challenge with the use of additives to reduce reactants likeasparagine is moisture migration that occurs during frozen storage withthe resulting return of asparagine to the surface and increasedacrylamide. Finally, the blackening that occurs after potatoes arebruised affects quality and recovery in processing French fries andchips. Damaged and bruised potatoes must be trimmed or are rejectedbefore processing, resulting in quality challenges or economic loss.

The “native technology” strategy of the present invention addresses theneed of the potato industry to improve the agronomic characteristics andnutritional value of potatoes by reducing the expression of polyphenoloxidase-5 (PPO-5), which is responsible for black spot bruise, theexpression of asparagine synthetase-1 (Asn-1), which is responsible forthe accumulation of asparagine, a precursor in acrylamide formation,and/or the expression of phosphorylase-L and kinase-R1, which areenzymes associated with the accumulation of reducing sugars thatnormally react with amino acids, such as asparagine, and form toxicMaillard products, including acrylamide. The partial or completesilencing of these genes in tubers decreases the potential to produceacrylamide. Use of the native technology of the invention allows for theincorporation of desirable traits into the genome of commerciallyvaluable potato plant varieties by transforming the potatoes only with“native” genetic material, that is genetic material obtained from potatoplants or plants that are sexually-compatible with potato plants, thatcontains only non-coding regulatory regions, without the integration ofany foreign genetic material into the plant's genome. Desirable traitsinclude high tolerance to impact-induced black spot bruise, reducedformation of the acrylamide precursor asparagine and reducedaccumulation of reducing sugars, with consequent decrease inaccumulation of toxic Maillard products, including acrylamide, improvedquality and food color control. The incorporation of these desirabletraits into existing potato varieties is impossible to achieve throughtraditional breeding because potato is tetraploid, highly heterozygousand sensitive to inbreeding depression.

The non-coding potato plant DNA insert sequences used in the presentinvention are native to the potato plant genome and do not contain anyAgrobacterium DNA. The DNA insert preferably comprises two expressioncassettes and is inserted into a transformation vector referred to asthe pSIM1278 transformation vector. The first cassette comprisesfragments of both the asparagine synthetase-1 gene (Asn1) and thepolyphenol oxidase-5 gene (Ppo5), arranged as inverted repeats betweenthe Agp promoter of the ADP glucose pyrophosphorylase gene (Agp) and theGbss promoter of the granule-bound synthase gene (Gbss). These promotersare predominantly active in tubers. The function of the second cassetteis to silence the promoters of the starch associated gene dikinase-R1(R1) and the phosphorylase-L gene (PhL). This cassette is comprised offragments of the promoters of the starch associated gene dikinase-R1(R1) and the phosphorylase-L gene (PhL), operably linked to the same Agpand Gbss promoters as the first cassette. These expression cassettescontain no foreign DNA, and consist of DNA only from either the selectedplant species or from a plant that is sexually compatible with theselected plant species.

The commercially valuable potato plant variety used in the presentinvention is Russet Burbank. Luther Burbank developed this variety inthe early 1870s. Plants are vigorous and have an indeterminate type ofgrowth. Stems are thick, prominently angled and finely mottled. Leafletsare long to medium in width and light to medium green in color. Theblossoms are few, white and not fertile. The cultivar is tolerant tocommon scab but is susceptible to Fusarium and Verticillium wilts,leafroll and net necrosis, and viruses. Plants require conditions ofhigh and uniform soil moisture and controlled nitrogen fertility toproduce tubers free from knobs, pointed ends and dumbbells. Jelly-endand sugar-end develop in tubers when plants are subjected to stress. Thetubers produced are large brown-skinned and white-fleshed, display goodlong-term storage characteristics, and represent the standard forexcellent baking and processing quality. Russett Burbank varieties havea high susceptibility to develop black spot bruise and also have highfree asparagine content and high senescence sweetening potential (Am. J.Potato Res (1966) 43: 305-314). The variety is sterile and widely grownin the Northwest and Midwest, especially for the production of Frenchfries.

The present invention provides a potato variety of significant marketvalue—namely Russet Burbank—transformed with the transformation vectorpSIM1278, identified using the polymerase chain reaction rather thanmarkers, and successfully propagated. Also provided are food productsmade from the tubers of the potato plant variety E12 of the presentinvention. Potato cultivar E12 has the following unique plant varietyidentifier with the Organization for Economic Cooperation andDevelopment (OECD): SPS-ØØE12-8.

Targeted gene silencing with native DNA reduces the level of the RNAtranscripts of the targeted genes in the tubers of the potato plantvariety E12. Asn1 and Ppo5 gene silencing is sufficient to significantlyreduce acrylamide formation by two to four fold without furtherinhibiting the starch associated genes kinase-R1 (R1) andphosphorylase-L (PhL). Thus, the tubers of the intragenic potato plantvariety E12 of the invention incorporate highly desirable traits,including a reduced ratio in free amide amino acids asparagine andglutamine, which is associated with reduced acrylamide formation uponfrying or baking Specifically, the potato variety E12 of the presentinvention is characterized by two- to more than four-fold reduction infree-asparagine content and a reduced discoloration associated withblack spot bruise. Furthermore, the potato variety E12 of the inventiondisplays a delay in the degradation of starch into the reducing sugarsglucose and fructose during storage. Impairment of starch-to-sugarconversion further reduces senescence sweetening and acrylamideformation and limits heat-induced browning.

Potato variety E12 of the present invention is therefore extremelyvaluable in the potato industry and food market, as its tubers producesignificantly less acrylamide upon heat processing and do not carry anypotentially harmful foreign genes.

EXAMPLES

The present invention uses native technology to integrate nativenon-coding DNA into the genome of selected potato plant varieties todevelop new intragenic potato plant varieties. The method includes traitidentification, design of vectors, incorporation of vectors intoAgrobacterium, selection of the recipient potato variety, planttransformation, evidence of absence of open reading frames, andconfirmation that the new potato plant varieties contain only the nativeDNA. The potato cultivar E12 of the present invention has a loweredpotential to form acrylamide, lower amounts of sucrose and is moreresistant to black spot bruise than its untransformed counterpart.

Example 1. The pSIM1278 Transformation Vector

The transformation vector pSIM1278 used in the invention was derivedfrom pSIM106, which was created by ligating a 0.4-kb potato plant DNAfragment (deposited as GenBank accession no. AY566555) with a 5.9-kbSacII-SphI fragment of pCAMBIA1301 (CAMBIA, Canberra, Australia),carrying bacterial origins of replication from plasmids pVS1 and pBR322,and the nptIII gene for bacterial resistance to kanamycin. An expressioncassette comprising the Agrobacterium ipt gene preceded by the Ubi-3promoter (Garbarino and Belknap, 1994) and followed by the Ubi-3terminator was introduced as a 2.6-kb SacII fragment into the vectorbackbone (Rommens et al., 2004). Insertion of the native 10-kb DNAsegment carrying two silencing cassettes into the DNA insert of pSIM106yielded pSIM1278. This vector was used for all transformations. ThepSIM1278 vector map is shown in FIG. 1. The vector backbone region is9,511 bp, as it starts at position 9,957 bp and ends at position 19,468bp. The backbone DNA consists mainly of bacterial DNA and providessupport maintenance of the DNA insert prior to plant transformation. Thebackbone portion is not transferred into the plant cells. The variouselements of the backbone are described in Table 1.

TABLE 1 Other Genbank Start-End effects on Accession Point in GeneticElement Origin Intended Function plant Number pSIM1278 Reference Patpromoter (pPat) Solanum Drives expression of the ipt None HM43928617,479- Unpublished including the coding tuberosum var. backbone markergene 19,2178 sequence for a 76- Ranger Russet amino-acid potatoubiquitin monomer Isopentenyl transferase Agrobacterium condensation ofAMP and Cytokinin NC_002377.1 16,744- Smigocki (ipt) gene tumefaciensisopentenylpyrophosphate formation 17,466 and Owens, to formisopentenyl-AMP, 1988 a cytokinin Terminator of the Solanum Terminateipt gene None GP755544.1 16,038- Garbarino ubiquitin-3 gene tuberosumtranscription 16,392 and Belknap, (tUbi3) 1994 Neomycin E. coliAminoglycoside None FJ362602.1 15,048- Courvalin et phosphotransferaseIII phosphotransferase 15,842 al., 1977 (nptIII) gene Origin ofreplication for E. coli Start position for plasmid None J01784.1 14,477-pBR322 (pBR322 ori) replication in bacterial cells 14,757 (pBR322 bom)E. coli pBR322 region for None J01749.1 14,077- replication in E. coli14,337 pVS1 replicon Pseudomonas pVS1 region for replication NoneAJ537514.1 12,667- (pVS1Rep) fluorescens in Agrobacterium (4,501-5,501)13,667 plasmid pVS1 pVS1 partitioning Pseudomonas pVS1 stability NoneAJ537514.1 11,074- protein StaA (PVS1 fluorescens (6,095-7,095) 12,074Stat) plasmid pVS1 overdrive Agrobacterium Enhances cleavage at the NoneK00549.1 9,963- tumefaciens Right Border site (103-132) 9,992

Example 2. The Plant DNA Insert and its Open Reading Frames (ORFs)

The DNA insert region used in the pSIM1278 is 10,147 bp long, from19,469 bp to 19,660 bp and from 1 bp to 9,956 bp. The DNA insertconsists of native DNA only and is stably integrated into the potatogenome. The DNA insert or a functional part thereof, is the only geneticmaterial of vector pSIM1278 that is integrated in the potato plantvarieties of the invention. The DNA insert is described in FIG. 2 andTable 2 below.

TABLE 2 Genbank Start-End Accession Point in Genetic Element OriginIntended Function Number pSIM1278 Reference Left Border (LB) siteSynthetic Site for secondary cleavage to AY566555 19,469-19,493 vanHaaren release single-stranded from (bases 1-25) et al. 1989 pSIM1278DNA flanking the LB sequence S. tuberosum Supports secondary cleavageAY566555 19,494-19,655 var. Ranger at LB (bases 26-187) Russet KpnIrestriction site S. tuberosum Site for connection of AF393847.119,656-1    transfer-DNA with LB flanking sequence. Promoter for the ADPglucose S. tuberosum One of the two convergent HM363752    2-2,261pyrophosphorylase gene var. Ranger promoters that drives (pAgp), 1stcopy Russet expression of an inverted repeat containing fragments ofAsn1 and Ppo5, especially in tubers Fragment of the asparagine S.tuberosum Generates with (9) double HM363759 2,262-2,666 Chawla etsynthetase-1 (Asn1) gene (1st var. Ranger stranded RNA that triggers al.2012 copy antisense orientation) Russet the degradation of Asn1transcripts to impair asparagine formation 3′-untranslated sequence ofthe S. verrucosum Generates with (8) double HM363754 2,667-2,810polyphenol oxidase-5 gene stranded RNA that triggers (Ppo5) (1st copy,in antisense the degradation of Ppo5 orientation) transcripts to blockblack spot bruise development Xba1 restriction site S. tuberosum Cloningsite. U26831.1 2,811-2,816 Spacer-1 S. tuberosum Sequence between the1st HM363753 2,817-2,973 var. Ranger inverted repeats Russet3′-untranslated sequence of the S. verrucosum Generates (6) doublestranded HM363754 2,974-3,117 polyphenol oxidase-5 gene RNA thattriggers the (Ppo5) (2nd copy, in sense degradation of Ppo5 orientation)transcripts to block black spot bruise development Fragment of theasparagine S. tuberosum Generates with (5) double HM363759 3,118-3,523Chawla et synthetase-1 (Asn1) gene (2nd var. Ranger stranded RNA thattriggers al. 2012 copy, in sense orientation) Russet the degradation ofAsn1 transcripts to impair asparagine formation EcoR1 restriction siteS. tuberosum Cloning site AY027522 3,524-3,529 Promoter for thegranule-bound S. tuberosum One of the two convergent HM3637553,530-4,215 starch synthase (pGbss) gene var. Ranger promoters thatdrives (1st copy, convergent Russet expression of an invertedorientation relative to the 1st repeat containing fragments copy ofpAgp) of Asn1 and Ppo5, especially in tubers Spe1 restriction site S.tuberosum Cloning site AY341425 4,216-4,231 pAgp, 2nd copy S. tuberosumOne of the two convergent HM363752 4,232-6,491 var. Ranger promotersthat drives Russet expression of an inverted repeat containing fragmentsof the promoters of PhL and R1, especially in tubers Fragment ofpromoter for the S. tuberosum Generates with (16) double HM3637586,492-7,000 potato phosphorylase-L (pPhL) var. Ranger stranded RNA thattriggers gene (1st copy, in antisense Russet the degradation of PhLorientation) transcripts to limit the formation of reducing sugarsthrough starch degradation Fragment of promoter for the S. tuberosumGenerates with (15) double HM363757 7,001-7,532 potato R1 gene (pR1)(1st var. Ranger stranded RNA that triggers copy, in antisenseorientation) Russet the degradation of R1 transcripts to limit theformation of reducing sugars through starch degradation Pst1 restrictionsite S. tuberosum Cloning site AY341425 7,533-7,538 Spacer-2 S.tuberosum Sequence between the 2nd HM363756 7,539-7,796 var. Rangerinverted repeat Russet

The DNA insert described in Table 2 that was used to create potato lineE12 of the present invention does not activate adjacent genes and doesnot adversely affect the phenotype of potato plant variety E12. Inaddition, the potato plant variety E12 of the invention does not producenovel proteins associated with open reading frames encoded by the DNAinsert.

Example 3. The Agrobacterium Strain and Transfection

The C58-derived Agrobacterium strain AGL1 was developed by preciselydeleting the transfer DNA of the hyper-virulent plasmid pTiBo542 (Lazoet al., 1991). A transposon insertion in the general recombination gene(recA) stabilizes recombinant plasmid vectors such as pSIM1278 (FIG. 1).AGL1 displays resistance against carbenicillin and rifampicin, and iseliminated from transformed potato tissue using timentin.

Stock plants of the Russett Burbank variety were maintained in magentaboxes with 40 ml half-strength M516 medium containing 3% sucrose and 2g/l gelrite (propagation medium). Potato internode segments of four tosix mm were cut from four-week old plants, infected with theAgrobacterium AGL1 strain carrying pSIM1278, and transferred to tissueculture media containing 3% sucrose and 6 g/l agar (co-cultivationmedium). Infected explants were transferred, after two days, to M404medium containing 3% sucrose, 6 g/l agar and 150 mg/1 timentin toeliminate Agrobacterium (hormone-free medium). Details of the methodsare described in Richael et al. (2008).

After one month, the infected explants were transferred to fresh mediumlacking any synthetic hormones and incubated in a Percival growthchamber under a 16 hr photoperiod at 24° C. where they started to formshoots. Many shoots expressed the ipt gene and displayed a cytokininoverproduction phenotype; these shoots were not considered for furtheranalyses. PCR genotyping demonstrated that about 0.3 to 1.5% of theremaining shoots contained at least part of the P-DNA while lacking theipt gene. Thus, no markers were used to select for the transformedplants. Details on ipt-based marker-free plant transformation werepublished by Richael et al. (2008).

The process of eliminating Agrobacterium started two days after explantinfection. For this purpose, tissues were subjected to the antibiotictimentin (150 mg/L) until proven to be free of live Agrobacterium. Proofwas obtained by incubating stem fragments of transformed events onnutrient broth-yeast extract (NBY medium) for 2 weeks at 28° C.(repeated twice). In accordance with 97 CFR Part 340, transformed plantswere transported and planted in the field only when free of liveAgrobacterium.

Potato plant variety E12 was analyzed by DNA gel blot analyses todetermine the structure and copy number of integrated DNA insertsequences and to confirm the absence of vector backbone sequences. Inaddition, molecular characterization was used to determine the sequenceof the junctions flanking the DNA insert and show stability of theinserted DNA. Sequencing information of the junctions provided the basisfor developing specific PCR tests for the intragenic potato plantvariety E12. Potato cultivar E12 was found to contain a single completecopy of the DNA insert as deduced from hybridization results whenvarious DNA digests were hybridized with AGP, ASN, PHL and GBS molecularprobes.

Example 4. Evidence for the Absence of the Vector Backbone DNA

Unlike many commercial transgenic crops, potato cultivar E12 of theinvention was confirmed to be free of Agrobacterium-derived DNAsequences that are used for transformation, such as vector backbone DNA,by three different methods: 1) First, the presence or absence of thenegative selectable isopentenyl isomerase (ipt) marker gene in thevector backbone was determined, as inadvertent transfer of backbone DNAcomprising the ipt gene expression cassette from Agrobacterium to plantcells would trigger ipt gene expression and, consequently, the formationof the cytokinin-type hormone isopentenyladenosine, 2) Southern blothybridization was then used on the transformed potato plants that hadpassed the first screening method to confirm the absence of backboneDNA, and 3) PCR was then designed to amplify fragments indicative ofjunctions between border regions and flanking backbone DNA or regionswithin the backbone DNA that flank the DNA insert. The efficacy of themethod was confirmed by using pSIM1278 DNA as a positive control. Potatocultivar E12 of the present invention did not produce PCR bandsindicative of the presence of vector backbone DNA.

Example 5. Stability of the Inserted DNA

The stability of DNA inserts was evaluated in the original transformantsand again in propagated plant material using both DNA gel blothybridization and trait evaluation. These studies were carried out toensure that intragenic events expressed the incorporated traits in aconsistent and reliable manner. Instability might be triggered by rarerecombination events or could also be caused by methylation. Becausepotatoes are normally propagated clonally, standard assessments forsexually propagated crops were not directly applicable, and tubersrather than seeds were used to define subsequent generations. Results ofDNA blot hybridization demonstrate consistent bands were present inmultiple generations, thus indicating stability. Further evidence forstability was obtained by confirming trait efficacy in generations oneand two tuber seed.

DNA insert stability was demonstrated in the originally-transformedmaterial (G0) by extracting and evaluating DNA from leaves of plantsthat had been propagated in vitro and never planted in soil. Forgeneration-1 (G1) analyses, two propagated plants from each intragenicvariety and one plant from each control were planted in the greenhouse;one of the tubers harvested from each plant was planted to obtain leavesfrom G1 plants that were used to isolate DNA and evaluate the G1generation. Tubers from this generation were planted again, and leavesof the resulting G2 plants allowed a characterization of thatgeneration.

Hybridization of DNA isolated from the Russet Burbank potato cultivarE12 of the present invention with the GBS probe revealed two commonbands (7.2 and 8.0-kb) and hybridization with the AGP probe revealedthree bands (1.4, 4.2 and 4.9-kb) in all lanes. These bands areindicative of DNA fragments of the unmodified genome. The presence oftwo additional bands in the DNA isolated from all intragenic material,one 2.2-kb band indicative of an internal DNA insert fragment and theother one representing a DNA insert junction fragment (4.4-kb for E12with GBS, 2.15-kb for E12 with AGP), indicated that the inserts of theoriginal transformant (G0) remained stable into the first and secondvegetative generations G1 and G2.

The catechol assay for Ppo activity provided additional evidence for DNAinsert stability in E12 variety, as all E12 tubers remained uncolored(FIG. 3), whereas the untransformed control material was stained. Thisdemonstrated that the variety E12 had retained its ability to silencethe Ppo5 gene in G2 tubers, after two years of field trials. Preparationof the field trial material involved repeated tissue culture andvegetative generations without affecting the stability of the insertedgenetic material.

Example 6. Junction Analysis and Variety-Specific Detection

At least one DNA insert/flanking plant DNA junction was sequenced usingeither Adapter Ligation-Mediated PCR or Thermal Asymmetric InterlacedPCR. The junction sequence was used to design primers for potatocultivar E12, and these primers were applied for variety-specificPCR-based detection methods. The methods developed were used to monitorplants and tubers in field and storage to confirm the absence ofintragenic material in tubers or processed food, and to ensure thepurity of organic seed.

Example 7. Efficacy and Tissue-Specificity of Gene Silencing

Gene silencing methods were employed to lower the activity of the Asn1,Ppo5, PhL, and R1 native proteins, and transcript levels rather thanprotein amounts were evaluated to link new phenotypic traits to changesat the molecular level.

Since strong silencing of the Asn1 gene involved in ASN (asparagine)formation in leaves and stems might adversely affect growth, the Agppromoter and the Gbss promoter, which are tuber- and stolon-specificpromoters and are much less active in photosynthetically-active tissuesand roots, were used to drive gene silencing in tubers and stolons. Thetranscript levels of the four targeted genes in various tissues of plantvariety E12 and its untransformed counterpart were determined byNorthern blot analysis.

In tubers of untransformed controls, transcript levels were “high”(easily detectable by northern blot hybridization) for the Asn1, PhL,and R1 genes and “low” for the Ppo5 gene. A comparison of northern blotsindicated that the Asn1, Ppo5, and PhL were expressed at similar levelsin tubers from greenhouse and field. In contrast, the R1 gene wassilenced slightly more effectively in greenhouse-grown control tubersthan in tubers from the field.

Strongly reduced transcript levels for the Asn1 and Ppo5 genes in tubersof variety E12 were associated with low-acrylamide potential and reduceddiscoloration associated with black spot bruising.

Transcript levels for the PhL gene were partially reduced in the tubersof line E12. This change was linked to reduced amounts of glucose andfructose. R1 transcripts were partially reduced (“whispered”) in tubersof E12 to help limit the degradation of starch into sugars.

The Asn1, Ppo5 and R1 genes were expressed at low levels in stolons ofuntransformed plants. In contrast, high transcript levels in thecontrols were associated with the PhL gene.

The transcript levels for the Asn1 and Ppo5 genes were even lower instolons from Russet Burbank E12 greenhouse-grown plants than in thecontrol stolons. This modification was expected and is not linked to anyundesired or unanticipated new phenotype. Expression of the PhL gene wasalso down-regulated in the stolons of intragenic variety E12.

The amounts of transcript for the R1 gene were slightly reduced(“whispered”) in stolons of variety E12. This molecular changecontributed to the limited degradation of starch into sugars.

In leaf tissues, the transcript levels for the Asn1 gene were similarfor line E12 and its untransformed counterpart. The transcript levelsfor the Ppo5 gene expression were in all cases undetectable, whereas thelevels for the PhL gene were consistently high among the originalvariety Russett Burbank and the transformed derivative E12. Thetranscript levels for the R1 gene were unaltered in variety E12 whencompared to its control.

In stem tissues, Asn1 gene transcript levels were similar for event E12and its control. The transcript levels for the Ppo5 gene were reduced inthe variety E12 of the invention. PhL gene expression was very similarin line E12 and its control and R1 gene expression was not reduced.

In root tissue, transcript levels for both the Asn1 and Ppo5 genes werereduced in variety E12. These results indicated that the promoters usedto drive silencing are partially functional in underground tissues.

In floral tissue, the Asn1 gene transcript levels were lower in thevariety E12 of the invention than in the original variety RussettBurbank. Transcripts were not detectable for the Ppo5 gene andexpression levels of the PhL and R1 genes were similar to controls.

These results demonstrated that the expression levels of the Asn1 andPpo5 genes were down-regulated in tubers and stolons in potato varietyE12, and that the R1 and PhL genes were at least partially silenced intubers and stolons in variety E12. Silencing was most effective intubers and stolons.

The selected potato variety E12 was more strongly affected in theexpression levels of the Asn1 and Ppo5 genes than in those of the R1 andPhL genes. These results coincided with the inventor's intent of (1)preventing the formation of Ppo protein and free ASN to the greatestextent possible, and (2) only partially blocking the conversion ofstarch into glucose and fructose.

The occasional changes in transcript levels in tissues other than tubersand stolons demonstrated some leakiness of the tuber/stolon-specificpromoter. It is also possible that small RNAs produced in tubers throughexpression of the silencing cassettes migrate into other tissues,especially roots and stems (Molnar et al., 2010). In most cases ofaltered expression levels in tissues other than tubers and stolons, thedifferences were minor. A summary of the down-regulated transcriptlevels in specific tissues of intragenic potato cultivar E12 is shown inTable 3. In Table 3, A=Asn1, P=Ppo5, L=PhL, and R=R1. Underlined lettersin Table 3 indicate strongly (>2-fold) down-regulated gene expressionlevels; expression levels are otherwise down-regulated by less than2-fold.

TABLE 3 Variety Tubers Stolons Roots Stems Leaves Flowers E12 A P L R AP R A P P L R A

Example 8. Field Performance and Tuber Evaluation

The 2009, 2010, and 2011 trials were planted mechanically to facilitateharvests and ensure that intragenic potatoes were kept separate fromunmodified material. For the 2009 evaluations, “nuclear seed” minitubersfrom each event and the control varieties were used to plant four orfive single-row plots (20 minitubers/plot) whereby the plots wererandomly distributed within blocks across the field. This randomizedcomplete block design (RCB) is typical for the evaluation of new potatovarieties and events. The approach taken in 2010 and 2011 was to usethree random plots per event and control per site, also using the RCBdesign with the number or replications (plots per event) equal to thenumber of blocks. Each plot in 2010 consisted of three rows of 20 seedpieces each from first generation (G1) tubers produced in Cherry County,NE, in 2009 for Russet Burbank. The Russet Burbank seed for the 2011trials was second generation (G2) produced in Cherry County, NE in 2010.In all trials, the seed of the intragenic line was handled similar toits unmodified control. Field grown tubers are desirable over minitubersas seed because they generate more vigorous and uniform plants thatproduce higher tuber yield and quality.

Each plot was evaluated qualitatively, in some cases using standardizedmonitoring scales, for differential responses to insect, disease, andenvironmental stresses that were not induced artificially but mightoccur spontaneously during the growing season. Mid-season monitoring wasconducted 2009, 2010, and 2011 just prior to or during early row closureand flowering (June-July for most trails except for the Florida trials,which were evaluated in April), to assess plant vigor, leaf color, leafsize, leaf curl, disease symptoms (presence/absence), andinsect-associated plant damage. In 2011, specific insects, diseases, andabiotic stressors common to the growing region were evaluated. Diseaseand insect pressure are generally highest during the mid and lateseason, but the plants were monitored for symptoms caused by pathogensand insects from July to September (March through May for Floridatrials). Late season monitoring of vine maturity and diseases wasperformed once prior to vine killing, a process intended to ensure tubermaturation and late-season skin set. Vine killing is induced either bymowing or flailing the vines or by using approved herbicides such asReglone according to the manufacturer's recommendations (JR Johnson,Roseville, Minn.). At this time, plants were also assessed for diseasesymptoms and insect damage. In some cases when disease symptoms wereidentified, a sprout test was also conducted to confirm the findings.

Means, standard deviations, and 90% confidence intervals were calculatedusing JMP 9.0.2. A conventional varieties range was generated byobtaining the minimum and maximum mean values (year*location*entry) ofall conventional varieties included in the experiments. Allcharacteristics for the Russett varieties were analyzed in JMP 9.0.2 bycombining data from multiple years and locations.

Example 9. Potato Cultivar E12 Characterization Summary

Potato variety E12 addresses the need of the potato industry to improvequality by reducing expression of the enzyme responsible for black spotbruise and to reduce acrylamide through lowering the concentration ofthe reactants, namely asparagine and reducing sugars. Potato variety E12was transformed with nucleic acid sequences that are native to thepotato plant genome and does not contain foreign DNA, Agrobacterium DNA,viral markers or vector backbone sequences In addition, agronomicstudies were conducted to ensure that the events grew the same asconventional controls, with the exception of the characteristicsassociated with the trait

Agronomic Characteristics

Evaluations of agronomic characteristics of potato variety E12 event andcontrol grown in 2009, 2010, and 2011 are shown in Tables 4-7. Resultswere analyzed by statistical methods where possible. Overall the datasuggest that there are no major differences between the Russet Burbankcontrol and the E12 Russet Burbank event.

Table 4 shows the number of site years for each characteristic testedfor variety E12. The agronomic characteristics for E12 and the Burbankcontrol are shown in Table 5. No statistically significant differenceswere detected between E12 and the control for seven of the agronomiccharacteristics. Vine maturity rating data were not able to bestatistically compared and the observed value of E12 was outside thecombined conventional varieties range (3.65 vs. 3.00-3.50,respectively). A statistically significant difference was detectedbetween E12 and the control for plant vigor (3.38 vs. 3.08); however,the value of E12 was within the conventional varieties range. In Table5, the stems per plant data is from 2011 only; conventional varieties(ConV) range equals a range of mean values of conventional Ranger,Burbank and Atlantic varieties; NA means that a statistical comparisonwas not possible.

The yield and grading characteristics of E12 and the Burbank control areshown in Table 6. There were no statistically significant differencesdetected for total yield, specific gravity, high sugar, or sugar end.Statistical analysis was not possible for total internal defects but thevalues for E12 were within the conventional varieties range. Fourdifferences were detected between E12 and the control for the 4-6 oz.category (25.3 vs. 21.5), the 6-10 oz. category (33.1 vs. 30.3), the10-14 oz. category (12.6 vs. 16.2), and the >14 oz. category (8.9 vs.13.1). All values for E12 for these differences were within theconventional varieties range. In Table 6, the specific gravity data isfrom 2011 only; conventional varieties (ConV) range equals a range ofmean values of conventional Ranger and Burbank varieties; NA means thata statistical comparison was not possible.

The flower colors of E12 and the Burbank control are shown in Table 7.Both purple/mixed flowers and white flowers were observed in differentplots for each entry.

TABLE 4 Number of Site Years Characteristic Event E12 Early Emergence 4Final Emergence 13  Stems Per Plant 4 Plant Vigor 8-11 Foliage Color8-11 Leaflet Size 8-11 Leaflet Curl 8-11 Senescence 3 Vine MaturityRating 7 Flower Color 2-4  Total Yield 11   %4-6 oz. 9-10  %6-10 oz.9-10 %10-14 oz. 9-10  % > 14 oz. 9-10 Specific Gravity 9-10 High Sugar9-10 Sugar Ends 9-10 Total Internal Defects 9-10

TABLE 5 Commercial Character- Burbank Reference istic Statistic E12 CtrlRange Early Mean 0.807 0.804    7.0-100.0 Emergence SD 0.227 0.215 90%CI   70.0-91.5%   70.2-90.5% p-Value 0.9367 Final Mean 0.937 0.944  79.0-100.0 Emergence SD 0.088 0.079 90% CI   91.1-96.2%   92.1-96.7%p-Value 0.6269 Stems Per Mean 2.8 2.7 1.6-5 Plant SD 0.61 0.5 90% CI2.52-3.16 2.41-2.93 p-Value 0.22 Plant Vigor Mean 3.4 3.1  2.3-4.3 SD0.6 0.35 90% CI 3.22-3.55 2.98-3.17 p-Value 0.0096 Foliage Mean 3.2 3.12.3-4 Color SD 0.38 0.27 90% CI 3.05-3.26 3.00-3.15 p-Value 0.1812Leaflet Size Mean 3 2.9 2.0-3 SD 0.24 0.27 90% CI 2.95-3.08 2.85-3.00p-Value 0.0815 Leaflet Curl Mean 2.3 2.2 1.0-3 SD 0.95 0.99 90% CI2.05-2.56 1.96-2.50 p-Value 0.2051 Senescence Mean 49.4 53.9   8.7-91.7SD 36.27 36.04 90% CI 26.96-71.92 31.55-76.23 p-Value 0.1013 Vine Mean3.7 3.1  2.3-4.5 Maturity SD 0.9 0.31 Rating 90% CI 3.33-3.97 2.95-3.18p-Value NA

TABLE 6 Commercial Character- Burbank Reference istic Statistic E12 CtrlRange Total Yield Mean 53.2 56.8 26.0-107.7 SD 21.8 20.9 90% CI46.3-60.0 50.5-63.2 p-Value 0.2079  4-6 oz Mean 25.3 21.5 2.3-32.2 SD9.4 8.4 90% CI 21.4-29.1 18.1-25.0 p-Value 0.0061  6-10 oz Mean 33.130.3 8.8-51.6 SD 7.2 8 90% CI 30.1-36.1 27.0-33.6 p-Value 0.0434 10-14oz Mean 12.6 16.2 6.7-31.7 SD 6.6 7.3 90% CI  9.9-15.3 13.2-19.1 p-Value0.0431   >14 oz Mean 8.9 13.1 0.0-59.3 SD 8.9 9.9 90% CI  5.3-12.6 9.0-17.2 p-Value 0.0077 Specific Gravity Mean 1.082 1.084 1.071-1.112 SD 0.008 0.007 90% CI 1.079-1.085 1.081-1.087 p-Value 0.1048 High SugarMean 12.3 8.2 0.0-36.1 SD 27.7 13.9 90% CI  0.9-23.6  2.4-13.9 p-Value0.4455 Sugar Ends Mean 18 21.4 0.0-70.3 SD 15.4 21.4 90% CI 11.6-24.312.6-30.2 p-Value 0.3789 Total Internal Mean 0.3 0.3 0.0-0.7  Defects SD0.8 0.7 90% CI 0.0-0.7 0.0-0.7 p-Value NA

TABLE 7 Number of Plots Purple or White Entry Mixed Flowers Flowers E123 5 Burbank Ctrl 3 8

Based on the data presented in Tables 4-7 for potato cultivar E12, itcan be concluded that there are no major differences in agronomiccharacteristics, flower color, yield and grading, and ecologicalinteractions between the untransformed Russet Burbank variety and potatocultivar E12. Therefore, based on the multi-year data, the Ranger Russetvariety E12 poses no significant risk of persistence in the environmentas a result of weediness or plant pest potential.

Black Spot Bruise Tolerance

Black spot bruise is a discoloration affecting bruised tubers thatrepresents one of the most important quality issues for the potatoindustry. The condition is a result of leakage of polyphenol oxidase(Ppo) from damaged plastids into the cytoplasm. In the cytoplasm, theenzyme oxidizes polyphenol, which then form dark precipitants. One ofthe two silencing cassettes of the pSIM1278 DNA insert contains twocopies of a fragment of the Ppo5 gene from Solanum verrucosum positionedas inverted repeat between regulatory elements. Expression of thisinverted repeat triggers silencing of the potato Ppo5 gene andsignificantly reduces the incidence of black spot bruise.

Field-grown tubers of potato cultivar E12 and a control Russet Burbankvariety, which is susceptible to black spot bruise, were assayed in twoways for black spot bruise tolerance.

Tuber discoloration is caused by the activity of polyphenol oxidase,which oxidizes phenols including catechol to produce compounds thatrapidly polymerize to produce pigments. An indirect method to test forblack spot bruise tolerance is based on pipetting 1-ml catechol (25 mMin 50 mM MOPS, pH6.5) onto the cut surfaces of tubers and monitoring forthe Ppo-dependent development of a dark brown precipitate. ThePpo-dependent development of a dark brown precipitate was assessed after20 min. Tubers of Russet Burbank controls and variety E12 were harvestedfrom three different plants that grew in Canyon County, ID, in 2009. Theresults of the catechol assay for potato cultivar E12 are shown in FIG.3. As seen in FIG. 3, the Russet Burbank control turned dark brownindicating black spot bruise, whereas potato cultivar E12 did not turndark brown indicating that E12 is more resistant to black spot bruisethan the untransformed Russet Burbank control.

The second method of assaying for bruise tolerance was by treating ninetubers of each control and potato cultivar E12 harvested in 2009 fromCanyon County, ID, to 10 revolutions in a bruise barrel (rotating drum),incubating at room temperature for 3 days, followed by peelingmechanically, and then counting the number of both black spot bruise andlineshatter (shatter bruise) spots. Table 8 shows the results and arepresented as the means of the results of three experiments, and theerror bars shown represent the standard error of the mean. Significancewas determined using the Student's two-tailed t-test (p<0.05).

TABLE 8 No. of bruises Difference from control Variety per tuber(P-value) Russet Burbank control 0.50 ± 0.79 Russet Burbank E12 0.00 ±0.00 P = 0.0107

As shown in Table 8, the barrel bruise test indicated undetectable blackspot bruise levels in potato E12 of the present invention. These resultsshow that the tubers of variety E12 are more resistant to black spotbruise than the tubers of untransformed control Russet Burbank variety,and that the DNA insert of pSIM1278 results in reduced impact-induceddiscoloration associated with black spot bruising.

Asparagine and Acrylamide Levels

Silencing of the asparagine synthetase gene resulted in averagereductions of 74% free asparagine in potato variety E12. The lowerlevels of asparagine, which combines with reducing sugars in theMaillard reaction to form acrylamide, result in average reductions of67% acrylamide in fries. The differences in asparagine and acrylamidebetween the Russet Burbank control and potato E12 are shown in Table 9.Before testing for acrylamide, the control and E12 were made into Frenchfries. All results were from tubers analyzed near the time of harvest.

TABLE 9 Free Percent Percent Asparagine Reduction Acrylamide ReductionVariety (ppm) from Control (ppb) from Control Burbank 2421  0% 493.8  0%Control E12 618.2 74% 162.4 67%

Potato cultivar E12 is a Russet Burbank variety with improved qualitythat has reduced expression of the enzyme responsible for black spotbruise and reduced acrylamide levels.

FURTHER EMBODIMENTS OF THE INVENTION

The research leading to potato varieties which combine the advantageouscharacteristics referred to above is largely empirical. This researchrequires large investments of time, labor, and money. The development ofa potato cultivar can often take up to eight years or more fromgreenhouse to commercial usage. Breeding begins with careful selectionof superior parents to incorporate the most important characteristicsinto the progeny. Since all desired traits usually do not appear withjust one cross, breeding must be cumulative.

Present breeding techniques continue with the controlled pollination ofparental clones. Typically, pollen is collected in gelatin capsules forlater use in pollinating the female parents. Hybrid seeds are sown ingreenhouses and tubers are harvested and retained from thousands ofindividual seedlings. The next year one to four tubers from eachresulting seedling are planted in the field, where extreme caution isexercised to avoid the spread of virus and diseases. From thisfirst-year seedling crop, several “seed” tubers from each hybridindividual which survived the selection process are retained for thenext year's planting. After the second year, samples are taken fordensity measurements and fry tests to determine the suitability of thetubers for commercial usage. Plants which have survived the selectionprocess to this point are then planted at an expanded volume the thirdyear for a more comprehensive series of fry tests and densitydeterminations. At the fourth-year stage of development, survivingselections are subjected to field trials in several states to determinetheir adaptability to different growing conditions. Eventually, thevarieties having superior qualities are transferred to other farms andthe seed increased to commercial scale. Generally, by this time, eightor more years of planting, harvesting and testing have been invested inattempting to develop the new and improved potato cultivars.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedpotato plants, using transformation methods as described below toincorporate transgenes into the genetic material of the potato plant(s).

Traditional plant breeding typically relies on the random recombinationof plant chromosomes to create varieties that have new and improvedcharacteristics. According to standard, well-known techniques, genetic“expression cassettes,” comprising genes and regulatory elements, areinserted within the borders of Agrobacterium-isolated transfer DNAs(“T-DNAs”) and integrated into plant genomes. Agrobacterium-mediatedtransfer of T-DNA material typically comprises the following standardprocedures: (1) in vitro recombination of genetic elements, at least oneof which is of foreign origin, to produce an expression cassette forselection of transformation, (2) insertion of this expression cassette,often together with at least one other expression cassette containingforeign DNA, into a T-DNA region of a binary vector, which usuallyconsists of several hundreds of basepairs of Agrobacterium DNA flankedby T-DNA border sequences, (3) transfer of the sequences located betweenthe T-DNA borders, often accompanied with some or all of the additionalbinary vector sequences from Agrobacterium to the plant cell, and (4)selection of stably transformed plant cells that display a desiredtrait, such as an increase in yield, improved vigor, enhanced resistanceto diseases and insects, or greater ability to survive under stress.

Thus, genetic engineering methods rely on the introduction of foreign,not-indigenous nucleic acids, including regulatory elements such aspromoters and terminators, and genes that are involved in the expressionof a new trait or function as markers for identification and selectionof transformants, from viruses, bacteria and plants. Marker genes aretypically derived from bacterial sources and confer antibiotic orherbicide resistance. Classical breeding methods are laborious andtime-consuming, and new varieties typically display only relativelymodest improvements.

In the “anti-sense” technology, the sequence of native genes is invertedto silence the expression of the gene in transgenic plants. However, theinverted DNA usually contains new and uncharacterized open readingframes inserted between the promoter and the terminator that encodeforeign amino acid sequences that may be undesirable as they interferewith plant development and/or reduce their nutritional value.

Expression Vectors for Potato Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990) Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. USA 84:131 (1987),DeBlock et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Potato Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific”. A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inpotato. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in potato. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. USA 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inpotato or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in potato.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin potato. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in potato. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.USA 82:3320-3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J.4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993))or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker et al., Plant Mol. Biol. 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129(1989); Frontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a potato plant. In anotherpreferred embodiment, the biomass of interest is seed or tubers. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene(s) to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al. Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A gene conferring resistance to a pest, such as soybean cystnematode. See e.g., PCT Application WO 96/30517; PCT Application WO93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, Van Damme et al., Plant Molec. Biol.24:25 (1994), who disclose the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT application US93/06487 which teaches the use of avidin and avidin homologs aslarvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 (Scott et al.), which discloses the nucleotidesequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO 95/16776, whichdiscloses peptide derivatives of Tachyplesin which inhibit fungal plantpathogens, and PCT application WO 95/18855 which teaches syntheticantimicrobial peptides that confer disease resistance.

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virusand tobacco mosaic virus.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

T. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S. Current Biology, 5(2)(1995).

U. Antifungal genes. See Cornelissen and Melchers, Plant Physiol.,101:709-712 (1993); Parijs et al., Planta 183:258-264 (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998).

V. Genes that confer resistance to Phytophthora blight, such as the R1,R2, R3, R4 and other resistance genes. See, Naess, S. K., et. al.,(2000) Resistance to late blight in Solanum bulbocastanum is mapped tochromosome 8. Theor. Appl. Genet. 101: 697-704 and Li, X., et. al.,(1998) Autotetraploids and genetic mapping using common AFLP markers:the R2 allele conferring resistance to Phytophthora infestans mapped onpotato chromosome 4. Theor. Appl. Genet. 96: 1121-1128.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic acidsand cyclohexones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotidesequence of a form of EPSP which can confer glyphosate resistance. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a PAT gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology 7:61 (1989) describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2625 (1992).

B. Decreased phytate content—1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, for example, this could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

D. Elevated oleic acid via FAD-2 gene modification and/or decreasedlinolenic acid via FAD-3 gene modification. See U.S. Pat. Nos.6,063,947; 6,323,392; and international publication WO 93/11245.

4. Genes that Control Male Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Methods for Potato Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc. Boca Raton, 1993) pages67-88. In addition, expression vectors and in-vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer—Several methods of plant transformationcollectively referred to as direct gene transfer have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant surface of microprojectiles measuring 1 to 4μm. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987); Sanford, J.C., Trends Biotech. 6:299 (1988); Klein et al., Bio/Tech. 6:559-563(1988); Sanford, J. C. Physiol Plant 7:206 (1990); Klein et al.,Biotechnology 10:268 (1992). See also U.S. Pat. No. 5,015,580 (Christou,et al.), issued May 14, 1991 and U.S. Pat. No. 5,322,783 (Tomes, etal.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985); Christouet al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of potato target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety in orderto produce a new transgenic variety. Alternatively, a genetic trait thathas been engineered into a particular potato line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties that do not contain that gene. As used herein,“crossing” can refer to a simple X by Y cross or the process ofbackcrossing depending on the context.

Persons of ordinary skill in the art will recognize that when the termpotato plant is used in the context of the present invention, this alsoincludes derivative varieties that retain the essential distinguishingcharacteristics of E12, such as a gene converted plant of that varietyor a transgenic derivative having one or more value-added genesincorporated therein (such as herbicide or pest resistance).Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the variety. The term “backcrossing”as used herein refers to the repeated crossing 1, 2, 3, 4, 5, 6, 7, 8, 9or more times of a hybrid progeny back to the recurrent parents. Theparental potato plant which contributes the gene(s) for the one or moredesired characteristics is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental potato plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol. In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the gene(s) ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a potato plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the one or moregenes transferred from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more traits or characteristics in theoriginal variety. To accomplish this, one or more genes of the recurrentvariety are modified, substituted or supplemented with the desiredgene(s) from the nonrecurrent parent, while retaining essentially all ofthe rest of the desired genes, and therefore the desired physiologicaland morphological constitution of the original variety. The choice ofthe particular nonrecurrent parent will depend on the purpose of thebackcross. One of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered or added to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance, it may be necessary to introduce a testof the progeny to determine if the desired characteristic has beensuccessfully transferred.

Likewise, transgenes can be introduced into the plant using any of avariety of established recombinant methods well-known to persons skilledin the art, such as: Gressel, 1985, Biotechnologically ConferringHerbicide Resistance in Crops: The Present Realities, In Molecular Formand Function of the Plant Genome, L. van Vloten-Doting, (ed.), PlenumPress, New York; Huttner, S. L., et al., 1992, Revising Oversight ofGenetically Modified Plants, Bio/Technology; Klee, H., et al., 1989,Plant Gene Vectors and Genetic Transformation: Plant TransformationSystems Based on the use of Agrobacterium tumefaciens, Cell Culture andSomatic Cell Genetics of Plants; Koncz, C., et al., 1986, The Promoterof T_(L)-DNA Gene 5 Controls the Tissue-Specific Expression of ChimericGenes Carried by a Novel Type of Agrobacterium Binary Vector; Molecularand General Genetics; Lawson, C., et al., 1990, Engineering Resistanceto Mixed Virus Infection in a Commercial Potato Cultivar: Resistance toPotato Virus X and Potato Virus Y in Transgenic Russet Burbank,Bio/Technology; Mitsky, T. A., et al., 1996, Plants Resistant toInfection by PLRV. U.S. Pat. No. 5,510,253; Newell, C. A., et al., 1991,Agrobacterium-Mediated Transformation of Solanum tuberosum L. Cv. RussetBurbank, Plant Cell Reports; Perlak, F. J., et al., 1993, GeneticallyImproved Potatoes: Protection from Damage by Colorado Potato Beetles,Plant Molecular Biology; all of which are incorporated herein byreference for this purpose.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing and genetic engineering techniques. These traits may or maynot be transgenic; examples of these traits include but are not limitedto: herbicide resistance; resistance to bacterial, fungal or viraldisease; insect resistance; uniformity or increase in concentration ofstarch and other carbohydrates; enhanced nutritional quality; decreasein tendency of tuber to bruise; and decrease in the rate of starchconversion to sugars. These genes are generally inherited through thenucleus. Several of these traits are described in U.S. Pat. No.5,500,365, U.S. Pat. No. 5,387,756, U.S. Pat. No. 5,789,657, U.S. Pat.No. 5,503,999, U.S. Pat. No. 5,589,612, U.S. Pat. No. 5,510,253, U.S.Pat. No. 5,304,730, U.S. Pat. No. 5,382,429, U.S. Pat. No. 5,503,999,U.S. Pat. No. 5,648,249, U.S. Pat. No. 5,312,912, U.S. Pat. No.5,498,533, U.S. Pat. No. 5,276,268, U.S. Pat. No. 4,900,676, U.S. Pat.No. 5,633,434 and U.S. Pat. No. 4,970,168.

DEPOSIT INFORMATION

A tuber deposit of the J.R. Simplot Company proprietary POTATO CULTIVARE12 disclosed above and recited in the appended claims has been madewith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The date of deposit was May 23, 2013.The deposit of 25 vials of microtubers was taken from the same depositmaintained by J.R. Simplot Company since prior to the filing date ofthis application. All restrictions will be irrevocably removed upongranting of a patent, and the deposit is intended to meet all of therequirements of 37 C.F.R. §§1.801-1.809. The ATCC Accession Number isPTA-120372. The deposit will be maintained in the depository for aperiod of thirty years, or five years after the last request, or for theenforceable life of the patent, whichever is longer, and will bereplaced as necessary during that period. A deposit of the J.R. SimplotCompany proprietary plasmid, pSIM1278 disclosed herewith and recited inthe appended claims has been made with the American Type CultureCollectton (ATCC), 10801 University Boulevard, Manassas, Va. 20110. Thedate of deposit was Jul. 11, 2017. The deposit of 25 vials of plasmidswas taken from the same deposit maintained by J.R. Simplot Company sinceprior to the filing date of this application. All restrictions will beirrevocably removed upon granting of a patent, and the deposit isintended to meet all of the requirements of 37 C.F.R. §§1.801-1.809. TheATCC Accession Number is PTA-124287. The deposit will be maintained inthe depository for a period of thirty years, or five yeas after the lastrequest, or for the enforceable life of the patent, whichever is longer,and will be replaced as necessary during that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. The potato transformation vector pSIM1278,wherein said potato transformation vector was deposited under ATCCAccession No. PTA-124287.